Epicuticular waxes form the outermost layer of the aerial portion of the plant and are thus the first line of interaction between the plant and its environment. The physical properties of this wax layer protect the plant from numerous environmental stresses. For example, the hydrophobic nature of wax prevents dehydration (nonstomatal water loss) and aids in shedding rainwater. The reflective nature of wax protects the plant against UV radiation (Reicosky and Hanover, 1978). Waxes are also known to protect against acid rain (Percy and Baker, 1990) and, because they are a good solvent for organic pollutants, they are able to impede the uptake of aqueous foliar sprays (Schreiber and Schonherr, 1992). Furthermore, surface waxes protect plants from bacterial and fungal (Jenks et al., 1994) pathogens ad play a role in plant-insect interactions (Eigenbrode and Espelie, 1995). Recently it has been shown that some of the compounds found in epicuticular waxes are also present in the tryphine layer of pollen grains (Preuss et al., 1993). Without these compounds the tryphine layer erodes, resulting in pollen that is unable to function causing male sterility.
Epicuticular waxes are composed of long chain, hydrophobic compounds all derived from saturated very long chain fatty acids (VLCFAs), that are synthesized within and then secreted from the epidermis. VLCFAs are defined as those fatty acids whose chain length is 20 or more carbons long. The lengths will vary from plant to plant, but typically, the wax VLCFAs are approximately 26-34 carbon long. These VLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system by sequential additions of C2 moieties from malonyl-coenzyme A (CoA) to pre-existing fatty acids derived from the de novo fatty acid synthesis (FAS) pathway of the plastid. Analogous to de novo FAS it is thought that each cycle of FAE involves four enzymatic reactions; (1) condensation of malonyl-CoA with a log chain acyl-CoA, (2) reduction to .beta.-hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the elongated acyl-CoA (Fehling and Mukherjee, 1991). Together these four activities are termed the elongase (von Wettstein-Knowles, 1982). VLCFAs in the epidermis are then converted to the other wax components through a number of pathways consisting of multienzyme complexes. For example VLCFAs are converted to aldehydes by fatty acyl-CoA reductase (Kolattukudy, 1971). These aldehydes can either be reduced by aldehyde reductase to produce primary alcohols (Kolattukudy, 1971), or decarbonylated by an aldehyde decarbonylase to produce odd chained alkanes (Cheesbrough and Kolattukudy, 1984). Alkanes can then undergo oxidation to form firstly secondary alcohols and then ketones (for review see Post-Beittenmiller, 1996). Very little is known at the molecular level about the components that are involved in the biosynthesis of wax specific compounds and their secretion onto the plant surface. Genetic studies have shown that there are a large number of genes involved in these processes (for example, 22 loci have been reported in Arabidopsis, 84 in barley). However only a few of these genes have been isolated so far and the biochemical role of their gene products remains unknown (Lemieux, 1996).
In addition to being made in the epidermal cells, VLCFAs also accumulate in the seed oil of some plant species. To date, developing seeds have been the primary focus of research into VLCFA biosynthesis. In seeds VLCFAs are incorporated into triacylglyerols (TAGs), as in the Brassicaceae, or into wax esters, as in Jojoba. The seed VLCFAs include the agronomically important erucic acid (C22:1), with oils containing this fatty acid used in the manufacture of lubricants, nylon, cosmetics, pharmaceuticals and plasticisers (Battey et al., 1989); Johnston and Fritz, 1989). Conversely, VLCFAs have detrimental nutritional effects and are therefore undesirable in edible oils. This has led to the breeding of Canola rapeseed varieties that are almost devoid of VLCFAs (Stefansson et al., 1961).
The seeds of Arabidopsis contain approximately 28% [w/wt of total fatty acids (FA)] of VLCFAs, eicosenoic acid (20:1) being the predominant VLCFA (21% of wt/wt of total FA). To identify the gene products that are involved in the synthesis of seed VLCFAs and establish the VLCFA biosynthetic pathway, several groups performed mutational analysis and screened for seed that had reduced VLCFA content. Each group independently identified the FATTY ACID ELONGATION1 gene (FAE1; James and Dooner, 1990; Kunst et al., 1992; Lemieux et al., 1990). A mutation at this locus resulted in reduced VLCFA levels (&lt;1% wt/wt of total FA) in the seed. Several other mutations that were non-allelic to FAE1 were also isolated. However, these mutations had a less pronounced effect in that VLCFAs still constituted 6.7% (wt/wt of total FA) of the seed fatty acid (Katavic et al., 1995; Kunst et al., 1992). Thus, despite the fact that four enzymatic activities are required for each elongation step, the FAE1 gene was the only one found by mutant analysis that resulted in almost complete loss of VLCFA synthesis in the seed.
The Arabidopsis FAEI gene was subsequently cloned (James et al., 1995; WO 96/13582), and showed homology to three condensing enzymes: chalcone synthase, stilbene synthase and .beta.-ketoacyl-[acyl carrier protein] synthase III (17 amino acids were identical to a 50 amino acid region of a consensus sequence for condensing enzymes). Based on this homology it was proposed that FAE1 encodes a .beta.-ketoacyl-coenzyme A synthase (KCS), the condensing enzyme which catalyzes the first reaction of the microsomal fatty acid elongation system (James et al., 1995). As determined by Northern analysis, the FAE1 gene is expressed in seeds of Arabidopsis, but is absent from leaves (James et al., 1995). This result is consistent with the fact that the faeI mutation affects only the fatty acid composition of the developing seed, having no pleiotropic effects on fatty acid composition of the vegetative, or floral parts of the plant. Thus, FAE1 is regarded as a seed-specific condensing enzyme.
Recently a cDNA from Jojoba seeds involved in the syntheses of VLCFAs has been isolated (Lassner et al., 1996; WO 95/15387). The protein encoded by this cDNA showed high homology to FAE1 (52% amino acid identity), and biochemical analysis demonstrated that it has a KCS activity. Using Jojoba KCS cDNA, Lassner et al. (1996) were able to complement the mutation in a Canola variety of Brassica napus, restoring a low erucic acid rapeseed line to a line that contained higher levels of VLCFAs. This suggests that in Canola, the mutation is in the structural gene encoding KCS, or a gene affecting KCS activity. Thus, both in Arabidopsis and Brassica napus, the mutations that result in the abolition of VLCFA synthesis seem to affect the condensing enzyme.
If four enzyme activities are necessary for an elongation step, and FAE1 and Jojoba-KCS only encode the KCS activity, one might expect to find other complementation groups that result in very low levels of VLCFAs synthesis. Because these complementation groups were not found in mutation screenings, Millar and Kunst (1997) have hypothesized that these three activities are not seed specific, but ubiquitously present throughout the plant and shared with other FAE systems involved in VLCFA formation including wax biosynthesis. To test this FAE1 was ecotopically expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. Expression of FAE1 alone in these cells resulted in the biosynthesis and accumulation of VLCFAs. This demonstrated that the condensing enzyme is the pivotal control point of the elongase, controlling not only the amounts of VLCFAs produced, but also their chain lengths. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression, and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized in any given cell (Millar and Kunst, 1997).
Thus, considering the central role of the condensing enzyme for VLCFA synthesis, the isolation of genes encoding condensing enzymes involved in the production of wax specific VLCFAs would facilitate the modification of wax composition through genetic engineering. Furthermore, since the majority of wax components are derived from VLCFAs, the availability of such genes would offer the potential to modify the wax load itself. This offers the potential to modify the susceptibility of plants to environmental stresses such as ultraviolet light, heat and drought, as well as the ability of plants to withstand insects and pathogens. The present invention is directed towards nucleic acids that encode condensing enzymes for VLCFA synthesis.